Visualisation of tissues and cells

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Why is it important to visualise tissues and cells?
-allows scientists to discover cellular and molecular composition of tissues and cells
-without the ability to see tissues, we would not know different types and functions
-scientists discover cellular functions like cell divison
-diagnose diseases like s
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How can we visualise tissues and cells?
-anything smaller than 200mm is what we can see
-light microscope for:10mm-200nm
-electron microscope: 100Mm to 1nm
-fluorescence microscope from 10mm to 10nm
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What is a wavelength?
distance between two neighbouring waves
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How is resolution increased?
moving left to right increases resolution as waves come closer together. Shorter the wavelength the better the resolution
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What microscope has the best resolution?
Electron microscope
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What is magnification?
the apparent increase in size of an object
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What does a beam of radiation do when it passes through a lens?
it refracts
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What do glass lenses refract?
light
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What does magnetic lenses refract?
electron beams
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How is magnification done by a convex glass lens?
-Thickness of lens
-Curvature of lens
-Speed of light in the lens
it ends up with a enlarged inverted image where the focal point is where the two beams meet
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What is resolution?
it is the ability to distinguish between objects that are objects together. The smallest distance between 2 particles at which they can be seen as separate objects
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How is resolution distance measured?
0.61 x wavelength/ numerical aperture
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How many micro metres apart do particles have to be to be seen as separate?
1micrometre
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What is the maximum resolution of a light microscope?
0.2Mm
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What is the maximum resolution for an electron microscope?
0.0078nm
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What is numerical aperture?
is the ability of an object to collect light. If it can collect a lot of light, it will have a high AP. High AP allows best resolution distance
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What does immersion oil do?
Can increase resolution as it makes sure light does not refract
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What is contrast?
it is the differences in intensity between 2 objects, or between an object and its background
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What is contrast important for?
It is important in determining resolution
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How is contrast increased?
with staining
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What can light microscopes be used with?
live, unstained cells and fixed stained specimens
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What are the parts in order of a light microscope?
ocular lens
path of light
prism
objective lens
specimen
condenser lenses
illuminator
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How is total magnification found?
mag of objective lens x mag of ocular lens
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What are used with electron microscopes?
fixed and stained or coated samples with electron dense material (Gold)
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What are the 2 types of electron microscope?
Transmission electron microscope and scanning electron microscope
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What does TEM do?
electron beams pass through the sample so can see internal structures of the cell
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What does SEM do?
electrons are focused on the surface of the cell so the image would be of the surface of the cell
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Why do electron microscopes have shorter wavelengths of electrons?
They give a greater resolution up to 200 times better than a light microscope
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What do fluorescent substances do?
absorb UV light and emit visible light
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What are some cells stained with to be fluorescent?
Fluorophores
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What can fluorescent microscopes be used with?
fixed or live cells and tissue
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How are specimens prepared for microscopy?
-fixation of tissue
-embedding in a supporting medium
-sectioning into thin sections
-staining sections to enhance contrast
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What is used when the specimen is cut?
a microtone
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What are the two main mechanisms in fixation?
cross linking and coagulation
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What is cross linking?
covalent bond formation in proteins and between them which causes tissues to stiffen and resist degradation
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What does coagulation do?
caused by dehydration of proteins through the use on an organic solvent like alcohol to deform the protein- makes hydrophobic regions move to the surface
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What can a coagulant or fixation help to do?
help embedding media like paraffin wax to penetrate tissues.
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What is a common fixative?
formalin
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What is formalin dissolved in water?
formaldehyde
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What does formaldehyde do during fixation?
Attaches to primary means such as those found on side chains of amino acids lysine and glutamine to form a stable cross link called methylene bridge- slow and takes one/two days
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What needs to be considered before fixation?
the diffusivity
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What do fixatives do?
diffuse a distance through the tissues at a rate related to the coefficient of diffusion multiplied by the square root of time
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What happens once formalin reaches the centre of the tissue?
coagulation needs to occur so the sample size should be limited to 4mm thick for thorough fixation
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What needs to be considered after diffusivity in fixation?
the volume in pH of the fixative
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When does formalin work best?
When buffered with phosphate to maintain a neutral pH so excess acid is not produced which causes artefacts in tissue.
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What is embedding?
involves supporting specimens and media that has a similar mechanical rigidity to the specimen itself
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Why is choosing the right embedding media important?
as can cause deficits while sectioning if it is too stiff or weak.
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What is the most common embedding media?
paraffin wax
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What has to happen to the specimen before embedded in paraffin wax.
must be dehydrated by replacing water in the tissue with ethanol then xylenes, then warmed with paraffin wax.
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What happens once the wax has infiltrated the sample?
it is carefully positioned and surrounded with additional wax using a mould to form a block
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What are the samples attached to for sectioning?
a tissue cassette- cut thin slices of a sample from an embedding block
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How thick are the sections usually after sectioning?
4-10 microns thick for use with a light microscope
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How are the sections cut?
metal, glass or diamond blade put on a microtone and sample is placed in a sample holder. Sample then advanced to cutting surface and drawn across the blade to create a thin slice
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What is done with paraffin embedded samples after sliced?
placed in a warm water bath and then put on a slide to dry
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What do thin sections of specimens allow?
allows light or electrons to pass through so can help identify cellular details and helps improve access of dyes and antibodies
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What are the 3 types of staining methods?
-histology
-histochemistry
-immunohistochemistry
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What is histology?
Uses organic dyes with affinity for particular subcellular components. Basic dyes stain acidic structures and acid dyes stain alkaline structures
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What is histochemistry?
identification of macromolecules like DNA, RNA and chemical compounds like Ca+2 in cells and tissue using dyes
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What is immunohistochemistry?
uses antibodies to detect and localise specific proteins
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What are the most common histology methods?
Haematoxylin and eosin
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What is haematoxylin?
A basic dye and binds to acidic components like DNA and RNA. It stains the nuclei
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What is eosin?
An acidic dye binding to alkaline components like proteins and gives a pink stain in images. It stains the cytoplasm
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What histology staining is massons trichrome?
uses 3 organic dyes: aniline blue-stains collagen blue, biebrich scarlet which stains cytoplasm red and haematoxylin which stains nuclei dark blue
-used to identify and quantify fibrous tissue-collagen
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What is an example of histochemistry?
PAS-periodic acid schiff and stains carbs. Allows us to detect carb macromolecules like glycogen and mucosubstances like glycoproteins glycolipids in tissues
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What does periodic acid do?
oxidizes sugars and exposes aldehyde groups. Aldehyde groups react with schiff reagent to give a purple magenta colour. Goblet cells are what is stained in the duodenum. Hepatocytes stain with PAS too.
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What does immunohistochemistry do?
uses antibodies to detect specific proteins. Antibody antigen specific interactions occurs and antibodies are conjugated with fluorescent dyes.
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What are the two types of immunohistochemistry?
Direct-conjugated primary antibody
indirect- primary antibody and conjugated secondary antibody
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What can using 2 antibodies in indirect cause?
result in unspecific binding
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When should you only use indirect binding?
to amplify the signal when you want to express very low amounts of protein
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Card 2

Front

How can we visualise tissues and cells?

Back

-anything smaller than 200mm is what we can see
-light microscope for:10mm-200nm
-electron microscope: 100Mm to 1nm
-fluorescence microscope from 10mm to 10nm

Card 3

Front

What is a wavelength?

Back

Preview of the front of card 3

Card 4

Front

How is resolution increased?

Back

Preview of the front of card 4

Card 5

Front

What microscope has the best resolution?

Back

Preview of the front of card 5
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