Investigative techniques Flashcards
Identify 3 requirements for light microscopy.
Stain (H&E)
Paraffin
Formalin solution
What role does formalin solution have in light microscopy?
This preserves the specimen ensuring that no putrefaction occurs.
What role does paraffin have in light microscopy?
Embed the tissue in paraffin so that it is easier to cut thin slices to make it easier to see under a microscope as more light can pass through.
What does H&E stand for and what is its role in light microscopy/
Haematoxylin and Eosin stain
Stains the specimens so it is easier to differentiate between the different tissues and components.
Describe the process of producing a frozen section.
- Specimen is placed on metal disc and frozen -20 to -30ºC
- Frozen specimen cut with microtome in cryostat.
- Stain with H&E
Describe what you would see if you used these stains in a light microscopy:
- H&E
- Masson’s trichrome
- Periodic Acid-Schiff stain
H&E: stains acidic components (DNA) blue/purple and basic components (cytosol) red/pink
Masson's trichrome: red=keratin & muscle fibres blue/green= collagen & bone light red/pink= cytoplasm brown/black= nuclei
Periodic Acid-Schiff stain: stains anything with a sugar magenta
Pros and cons of frozen sections.
+ quicker to do
+preserves fats
- lower quality specimens
- poorer stain retention
- shorter lifespan
- can have ice damage
Outline the process of fluorescent microscopy.
- Specimen has fluorescent molecules bound to it.
- Fluorescent molecules emit light when exposed to specific radiation wavelength.
- You would see different colours for different components and cell types.
When is fluorescent microscopy most commonly used?
Used for differentiating heterogeneity of a cancer
Outline the process of confocal microscopy.
- Use a fluorescent dye
- Lasers are projected to specimen and excite the fluorescent molecules.
- A light is emitted through a pinhole screen to a detector.
- Only focused part of light reaches detector
- Creates very sharp image.
Pros of confocal microscopy.
- high quality image produced
- 3D images
- can be used for live specimens (time lapse video)
Outline the process of autoradiography.
- Live specimen injected with a radioactive marker
- Histological section is coated in an emulsion to see the molecules marker
What is ultrasound?
Use of soundwaves to produce an image based on how long it takes for echoes to return. More reflection (hyperechoic) = white Less reflection (hypoechoic) = black
Describe the process of performing an ultrasound.
- Transducer emits ultrasound waves towards the tissues
- Waves echo off the tissue.
- Based on the density and distance of structures, the reflection times will differ.
- A black and white image will be produced due to the reflected echoes being recorded.
Describe the use and aim of an ultrasound.
Most commonly used to view fetuses or any internal body structures to find/exclude a source of disease.
The use of ultrasound is quick and doesn’t involve any radiation so it is safer to use.
Outline the process involved in TEM.
- Fix, embed, stain, cut
- e- beam passes through specimen
- Transmitted beam = lighter image
- Absorbed/scattered beams = dark image
Make a relative comparison of electron & light microscopy in terms of:
- resolution
- magnification
Electron microscopy allows for a much higher resolution and magnification than light microscopy.
E = 400x light resolution
E = 250x light mag.
Outline the process involved in freeze fracture electron microscopy.
Used to look at the cell membrane components.
- Tissue frozen to -160ºC and fractured by hitting the specimen with a knife edge.
- Fracture line will go through the plasma membrane so the interior structure is exposed.
Outline the process involved with SEM.
- e- projected towards the specimen
- all e- reflected back to cathode ray tube
- depending on where e- are reflected will give you a 3D image of the surface of the specimen.
What is an MRI?
Non-invasive, non-destructive tool that relies on a magnetic field that produces transverse images of soft tissues.
How does an MRI work?
- H atoms spin in random directions in their own magnetic field
- MRI produces a magnetic field and the atoms line up north/south.
- Applied radio frequency pulse matches all the atoms’ direction of spin.
- When radio freq. turned off, the originally unmatched atoms will spin (relax) to their original direction and emits energy by doing that.
- Energy sends a signal to the computer which creates an image.
When you look at an MRI image, you can see different shades of black, grey and white. How are these different contrasts produced?
The contrasts are created due to the different relaxation times of the H atoms within the different tissues.
