Week 1 Flashcards
Definition of Imaging
The visual representation of an object, such as a body part or celestial body, for the purpose of medical diagnosis or data collection, using any of a variety of usually computerised techniques.
What is an example of a type of imaging method which is excluded from the ‘imaging’ definition?
A photograph because it does not have a computerised technique element
What is a type of more abstract imaging methods?
Visualisation of calcium distribution in neurons with calcium imaging
What is the definition of functional imaging?
The task of functional brain imaging is to identify regions and their temporal relationships associated with theperformance of a well-designed task
What differentiates functional imaging from non functional?
- Functional provides added information if change that is happenning to respond in response to the scientific aspect
- It is often investigating a change in response to a stimulus or experiment design to see the function
What is the smallest level of resolution?
Molecular
What is the main issue of having an imaging method whihc measures to the sub-millimere in spatial and millisecond in temporal resolution?
This would provide too much information that would take too long to analyse
Is there an ideal imaging methodology which encapsulates sub-millimetre spatial and millisecond temporal resolution?
No single method today achieves this
What is an important thing to consider when choosing an imaging method?
Need to proportionally pick the method which has the required need of resolution- there is no point using a mehtod wih extremely fast temporal resolution to measure the slow growing of cells for example
What is the definition of spatial resolution?
Is the ability to distinguish two separate objects that are positioned close to each other.
What are 3 factors that influence the spatial resolution?
- Optics
- Contrast within the images
- Blurring in the imaged tissue (point spread function)
How can you improve the contrast of something we are imaging?
Use a stain
How would blurring affect spatial resolution?
The image becomes hazy and therefore bigger so there is less intensity in the profile mapped out
(same happens when there is less contrast)
What type of relationship is there between electromagnetic properties and resolution?
Linearly to the wavelength used
Why is there a limit to imaging using electromagnetic waves?
Because of diffraction there is a theoretical limit to the spatial resolution
What are three examples of Electro-magnetic imaging?
- X-ray
- Light microscope
- Electron microscope
What is the linear equation of theoretical resolution and EM imaging?
x= λ/sin(α)
Where:
* x: Minimum distance at which two objects can be resolved (resolution).
- λ: Wavelength of the imaging source (e.g., electron wavelength in EM).
- sin(α): Sine of the aperture angle (𝛼).
What is the aperture angle?
Angle of the object going into the objective
Is a larger or smaller angle aperture required for higher resolution?
Larger (inverse to the distance)
Is a larger or smaller minimum distance objects can be resolved required for a higher resolution
Smaller
What are two tricks to improve the theoretical resolution?
- Immersion lenses
- Super-resolution microscopy
How many times better is an electron microscope compared to a light one?
10,000 times
What is the signal to noise ratio (SNR)?
- Describes the relationship between the size of the meaningful signal and the size of the recorded noise
- It is a measure of the statistical accuracyof data from an imaging device
What is the SNR in imaging described by?
Pixel Example
The mean value in a pixel compared to the standard deviation of that pixel in repeated measurements (takes the same picture again and again to see if there are changes due to noise)
What are 4 sources of noise in light imaging?
- Photon shot noise
- Dark noise or sensor noise
- Biological motion
- Technical artefacts
What is Photon shot noise?
Is produced by the stochastic process at which photons are emitted from a light source (sensitive instruments pick up the varying intensity in light sources)
What can be used to overcome photon shot noise?
Lasers
What is dark noise or sensor noise?
Is the variation in the sensor read out in complete darkness. Basically all electronics have noise and variation (taking a pic i the dark- will portray grey and white dots or noise instead of the true blackness-because of a contrast issue)
What is biological motion?
Like a heart beat or respiration- the heart can be difficult to measure as it changes over time (could take the image at the same phase of the heart beat to get around this).
What are technical artefacts?
It is something that is seen on an image as abnormal but does not represent a real abnormality.
E.g., the susceptability artefact in fMRI.
How can you overcome bad SNR?
The basic idea behind improving the SNR is that the noise in your data is random and the signal you are interested in is not
Therefore if you record a signal several times and average you should end up with the mean of the real data whereas the noise cancels out
What types of imaging cannot be repeated to improve the SNR?
Invasive techniques such as PET scans.
What can invasiveness mean?
- Exposing the surface of the brain like in Optical Imaging.
- But it can also mean exposure to other damaging effects like radioactive material in PET
Why was light microscopy such an exciting scientific development?
Allowed a step change is the understanding of disease/medicine and research progression
Where does the word microscope originate from?
- Skopein– see
- Mikros– Little
What is a light microscope?
A microscope (device to magnify small objects) in which objects are lit directly by white light
What generation of people were the first to understand lenses?
The ancient Egyptians
What did the ancient Egyptions use in relation to lenses?
Used convex lenses to magnify small objects
Who invented the compound microscope?
Hands and Zacharius Jansen in 1591
What is a compound microscope?
Using a set of two lenses arranged in a way where the focal point of one lens falls on the focal point of another giving mroe magnification
Who published micrographia and what did it entail?
- 1665, Robert Hooke
- How to use mirror angles in a way to allow sunlight to enter the specimen
Who increased the curvature of the lens in order to increase the magnification of the light microscope and what did this lead to the discovery of?
- 1673, Anthony Van Leewenhoek
- Used the curvature to influence where the focal point was in order to discover- bacteria, water, sperm and blood cells
When looking through a light microscope are we looking at the image of a specimen or the specimen itself?
Image
What is magnification?
The ratio of the apparent size of the object as seen through the microscope (image size) to the real size of the object (object size)
Thus, an image being seen at a magnification of 100X means that its linear dimensions are 100 times those of the object giving rise to the image
How do you work out the total compound of the magnification?
- Objective x Ocular
- 100x * 10x = 1000x
What is the magnification of a single lens microscope ranging from?
- 1.5-2
- Mild magnification
What happens to the magnification when we add a second (compounded lens)?
- The focus point of the first image is arranged on the second image. Therefore there is the same effect of the first lens on the origional image ut again.
- Therefore we get double the magnification
What extra lens needs to be taken into account when using a compound microscope?
- Regarded as two converging lenses but..
- We also have the lens in our eye
- SO, the magnification of the first lens, second lens in the eye piece and the eye lens
What is resolution?
The ability of the microscope to distinguish seperate and distinct objects that lie in close proximity under a particular condition of usage
What is necessary in order to receive a clear resolution from a light microscope?
eye
- In order to percieve two object as seperate things. they have to fall onto two different retinal cells in the retina
- If the image is not small enough to fall onto two seperate cells, we cannot distinguish between the two object
How can we overcome the problem of bad resolution (minor) in a top down manner?
- If we know the cells are round then even if we see a cell that looks like 8
- We can understand that the scene is not fully resolved and assume that they are two seperate cells as this shape is not probable
What is numerical aperture?
Is a quantitative value for the angular aperure of the microscope lens, Ernst Karl Abbe (1840-1905)
What is the refractive index (n) and how is this important for numerical aperture?
- The refractive index (n) measures how light slows in a medium.
- Air between the cover slip does not have a high index
- So, we can use immersion (a drop of oil) which improves the refractive index- artificially improving the resolution
What is the equation for numerical aperture?
NA=n⋅sin(α)
Where:
* NA: Numerical aperture, a measure of the light-gathering ability of an optical system.
* n: Refractive index of the medium between the lens and the specimen (e.g., air = 1.0, water = 1.33, oil = 1.51).
𝛼
* α: Half the angle of the maximum cone of light that enters the objective lens (the acceptance angle).
What does a condensor add to a compound microscope?
Allows to increase and decrease the field of illumination and the light path
How do expensive and cheap compound microscopes differ in terms of their objective lens?
- In cheap microscopes when change the objective lens magnification, the focal point changes
- In expensive microscopes, there is the same focal point in different magnifications so don’t have to re-search for the cell when changing the magnification
Try and draw a compound microscope and label it
Labels-
- Condensor
- Illuminator
- Ocular lens
- Objective lens
- Stage with aperture
- Focus knobs
What are 5 types of light microscopy?
- Bright field
- Dark field
- Phase contrast
- Normarski differential interferene microscopy
- Flourescence
Describe Bright-field light micrscopy
- Entire field is illuminated by the condensor lens
- Specimens must be translucent and stained to provide a contrast between structures (once cut perfuse tissue down to 20micrometers cannot tell the differene between cells and nucleus as transparent)
What is the most common type of light microscopy?
Bright-field
Explain dark-field light microscopy
- Uses a special condensor lens to increase the illumination by indirectly illuminating the sample causing scattering into the lens
- Enhances the contrast on unstained specimens
- Allow visualisation of living specimens
Explain the mechanism of a dark-field light microscope
- Dark field condensor has a black disc in the centre
- Means that if there is no specimen on the stage then the light of illumination does not enter the objective
- If there is a sample on the stage, the tissue has a denser reflective index than air and scatters the light
- This then enters the objective and becomes visible
Describe a Phase contrast light microscope
- Uses a change in the phase rather than change in the light path
- lens system produces visible images from transparent objects
- Based on the principle that light changes its speed when passing through structures with different refractive indexes so that the structures appear lighter or darker than each other
- Without a change in the phase of light, the specimen is not visible
- allows visualisation of live, unstained specimens
Explain Normarski Differential Interference Contrast (DIC) light microscopy.
- Related method to the phase contrast - it relies on differences in the refractive index of components in a sample to generate contrast.
- Instead of phase shifts, DIC uses polarized light and optical gradients.
- Produces images that appear 3D but this is an illusion as it is simply a flat sample that has been cut
- High contrast and resolution as 3D appearance helps to tell the difference between components of the cell by increasing the features.
Explain flourescent light microscopy
- Some specimens that are exposed to a light of certain wavelength emit a light of a longer wavelength
- It has a strong UV light and filters that select rays of different wavelengths
What are the 6 steps of preparation for light miscroscopy?
- Fixation
- Dehydration and clearing
- Embedding
- Sectioning
- Mounting
- Staining
Some methods change the order
What is the purpose of fixation in light microscopy?
Prevents tissue degeneration and preserves structure
Explain immersion fixation
Submerge the sample in the chemical solution
What two chemicals are frequently used in perfusion and immersion fixation?
- Formaldehyde
- Paraformaldehyde
What is perfusion as a method of fixation?
Connect a needle to the aorta of a sample and push the chemical through the system and it will be dispersed everywhere through the tissue
What are three limitations of immersion and perfusion fixation techniques?
- May cause changes in fine structure (dissolving of lipids and denaturing) therefore cannot carry out DNA analysis and genetics at a later date
- Staining artefacts
- Chemicals are carcinogenic
What are the positives and negatives of freezing as a fixation technique?
POSITIVES
- Faster than chemical fixation
- Avoids dissolving lipids and denaturing therefore can carry out DNA analysis and genetics
NEGATIVE
- Specimens are short lasting
What is the purpose of dehydration and clearing in light microscopy?
Prepare the chemically fixed material for infiltration with embedding material i order to replace the water with a different chemical or remove it all together
What are the 4 steps of dehydrating and clearing a specimen?
- Dehydrate with an ascending series of graded alcohols
- Replace alcohol with clearing agent using a series of clearing agent/alcohol mixtures
- Clearing agent miscible with both alcohol and embedding material
- Tissue infiltrated with embedding material
Why is dehydration of a sample done in a graded manner?
The tissue will warp and tare so need to slowly preserve
What is the usual embedding material when dehydrating and clearing a specimen?
Xylene
What are the limitations of dehydrating and clearing a sample?
- May degrade the tissue
- Dehydration can cause shrinkage
What is the purpose of embedding?
Makes the tissue firm to facilitate sectioning and prevent crushing during the process
not all tissue processing use this
What are the two embedding mediums?
- Paraffin wax
- Plastic resin
How does embedding work?
- Embed the specimen into the medium
- Cut thin slices
- Used when the tissue is super thin and its hard to get a cross section
- If do not use, the tissue would collapse
What is the purpose of sectioning in light microscopy?
Thin slices needed to allow light to penetrate tissues and form an image
How is paraffin embedded material sliced and what is used?
- Using a microtome to slice around 3-8micrometers
- Bead on the side tells the step change
- Push the blade through the sample and pull the slide back
- It goes back by the amount on the settings
What is used and how are frozen sections sliced?
- Frozen tissue sections are sliced using a cryostat, which freezes the sample on a cooling stage.
- A microtome blade cuts sections 10–30 micrometers thick.
- Proper temperature control is crucial: if too cold, the specimen can shatter; if too warm, it may tear.
What is the purpose of mounting?
To decrease damage to the specimen during microscoping examination
How are paraffin section mounted?
Are floated on a hot (100degreesC) water bath and then placed on a glass slide
How are frozen sections mounted?
Put directly onto the glass side
What does the cover slip achieve?
Attempts to preserve the slide
How long can specimens last at the end of the preparation procedure?
Depending on the histological preparation, these can last for decades
What is the purpose of staining?
Used to provide contrast to show tissue structure
Whhat is the most common used dye in light microscopy?
A combination of Haematoxylin and eosin (H&E)
What does Haematoxylin stain for?
Stains DNA and other acidic structures blue
What does Eosin stain for?
Stains cytoplasmic components and collagen pink
What does Masson’s Trichrome stain do?
Stains collagen green
What does Van Geison stain do?
Stains elastin black
When would Van Geison’s stain be used?
In skin sections
What does periodic acid-schiff (PAS) stain do?
Stains carbohydrate-rich molecules bright pink
In what year did Cajal and Golgi win the Nobel prize?
1906
What theory did Golgi propose?
Reticular theory- where the nerves form a continuous network
What type of stain did Golgi create?
Silver chromate stain which selectively stains the whole neuron
What percentage of cells are stained using silver chromate?
2%- if stained all, it would be black- allowed to look at the connections
What could the original golgi stain not accomplish?
Could not visualise individual structures
How did Cajal improve Golgi’s stain?
Made a double impregnation to make it more reliable and visualise structures.
(Just applied it twice).
What did Cajal discover when he performed the double impregnation of the silver chromate stain?
That myelinated cells were not seen as they could not absorb the stain
How did Cajal overcome the issue of myelinated cells in staining?
Used embryogenic method where used cells before the myelin was fully formed
What is an epitope?
Also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system
What is immunohistochemistry (IHC)?
Refers to the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies bindingspecifically to the antigen
What is the most common IHC dye used?
Glial fibrillary acidic protein
Why is IHC helpful for for seeing glia vs neurons?
- They are difficult to determine under a light microscope or using a generic stain due to their close proximity
- Antibodies are so specific that it can attach to one cell type and then to a different one
- The cells are then dyed different colours
How does an IHC work and what is it useful to see?
- Uses tagged compounds that specifically interact with the molecule of interest
- Can see the distribution and localisation of proteins i.e. receptors, neurotransmitters, transporters, ion channels
What are the most common antibody tags?
- Non-fluorescent = peroxidase
- Fluorescent compounds
What is the general methodology of IHC?
- Tissue section incubated with a tagged antibody (specific for the antigen)
- Antibody binds to the antigen (protein)
- Tagged compound shows on microscopic examination
What does conjugated mean in terms of an IHC?
That the dye is attahed to the target protein
What is a common non-fluorescent marker used in hospitals for acute liver injuries?
Cytokeratin 18
What is an importent facet of antibody usage in IHC related to species?
Antibodies must be specific to the protein in the relevant species
Some antibodies can cross-react with more than one species
What are the two most basic types of antibody?
- Polyclonal
- Monoclonal
How does a polyclonal antibody work?
Attaches to the protein at several different epitope sites
What is a bennefit of polyclonal antibodies?
Bright identification of the location of the protein
What are the 4 steps to polyclonal antibody production?
- Protein (antigen) is isolated from the species we are working with
- Protein is injected into an animal of another species. The immune system of the animal kicks in detecting the foreign invader to produce antibodies against it
- B-lymphocytes in the spleen and lymph nodes produce antibodies
Different groups (clones) of lymphocytes recognise different parts of the protein (epitopes) and each clone produces antibodies against that part
- Blood sample taken, mixture of polyclonal antibodies are isolated from the plasma. Each can bind to different regions of the protein
Why are larger animals preffered in polyclonal antibody productions?
Have to extract blood, smaller mammals such as a mouse can only take a small sample of blood
Usually use rabbits and goats
What are monoclonal antibodies?
Antibody that is attaching to one specific epitope on the target protein- it is much more specific as only stain the cell of interest
However, because the number of antibodies is smaller, the intensity of the staining is reduced
What are the 5 steps of monoclonal antibody production?
- Protein (antigen) is isolated from the species we are working with
- Protein injected into a different species such as a mouse (does not need to be a larger animal)
- B-lymphocytes in the spleen are activated
- Activated lymphocytes are isolated and placed in a culture with lymphocytic tumour cells to produce hybridoma cells
- The different hybridoma clones produce antibodies to different epitopes and are isolated and cultured seperately
What are the advantages of monoclonal antibody production?
- Highly specific
- Binds strongly to protein
- Less non-specific binding
What are the 5 steps to IHC preparation?
- Fixation
- Dehydration and clearing
- Embedding
- Sectioning
- Mounting
What is the purpose of deparaffiniasation in IHC?
Paraffin wax interferes with the ability of the antibodies to bind as the wax is covering the protein sites therefore must be removed
What chemical is used for deparaffiniasation in IHC and what does it do?
Xylene
Dissolves the layer of paraffin
What is the purpose of antigen retrieval in IHC?
When the tissue is preserved, the formalin forms Aldehyde cross-links that mask the antigen (epitope), antigen retrieval uncovers the epitope
What are 3 different methods of antigen retrieval in IHC?
- Heat induced epitope retrieval- put in a microwave for a certain amount of time to dissolve the cross-links
- Room temp epitope retrieval- Uses acids to break the cross-links (HCL/formic acid)
- Proteolytic induced epitope- Uses trypsin, pepsin to break the cross-links
What is the purpose of permeabilisation in IHC?
To make the cell wall permeable when identifying intracellular structures
What are two methods of permeabilisation in IHC?
- Triton X
- Methanol
What is the purpose blockage of non-specific binding in IHC?
Prevent unspecific immunoreactivity, making sure antibodies are not carrying out protein on protein binding (instead of desired epitope binding)
What is the method of blocking non-specific binding in IHC?
- Incubate with bovin serum albumin (BSA), milk/casein or serum (from antibody host
What should be done before blocking non-specific binding on desired sample in IHC?
Take a tissue section where there desired structure is not present and do a stain to see how severe the non-specific binding is (control)
What are the two antibody incubation methods?
- Direct
- Indirect
Before carrying out antibody incubation, what are 3 parameters you need to determine?
- Optimal concentration of the antibody
- Length of incubation
- Temperature
What is the direct method of IHC incubation?
- Antibodies are tagged with appropriate label (could be monoclonal or polyclonal)
- Section incubated with labelled antibody
- Antibody will bind specifically with antigen if present
- Unbound antibody washed away
- This leaves the antibodies on the cells in the locations we want to investigate
How intense is the dye in direct incubation?
Is low because there is only one epitope binding for each antibody
What is the indirect incubation method in IHC?
- Section incubated with unlabelled primary antibody (e.g. rabbit) specific to the epitope of the sample type we are interested in
- Primary antibody binds to antigen
- Excess antibody washed away
- Section incubated with labelled secondary antibody (e.g. goat)
- Secondary antibody binds to the primary antibody of the rabbit
How intense is the dye in indirect incubation IHC?
Intense because there are more dye molecules for each individual epitope site
What are the advantages of direct incubation in IHC?
- Quicker
- Less expensive
What are the advantages of indirect incubation in IHC?
- Signal is stronger, because multiple secondaries can recognise one primary
- More sensitive
In the indirect incubation method of IHC what are secondary antibodies often tagged with?
Peroxidase
What is the method of tagging secondary antibodies in an indirect incubation method of IHC?
- Slides are incubated with a peroxidase substrate- 3,3- diaminoazobenzidine (DAB)
- DAB is oxidised in the presence of peroxidase to form an insoluble brown precipitate
- The brown precipitate localises the antigen
