1. Intro and Investigative Techniques Flashcards
How does a Bright Field Microscope work? (3 steps)
1) Stain sample
2) Illuminated with white light from below
3) Observed from above
To view a tissue sample under a bright field microscope, what needs to be done first? What chemicals do you use for these steps? (3 points)
1) Preserve the tissue - Formalin prevents rotting
2) Embed the tissue - Melted paraffin allows thin slicing
3) Stain the tissue - Haematoxylin/Eosin so components can be seen
What does Haematoxylin stain most strongly?
Eosin? Both?
1) The Nucleus
2) Cytoplasm and Extracellular matrix
3) Nucleus seen more clearly and more detail in cytoplasm
An alternative to using Melted Paraffin is the Frozen Section. Briefly, how is this done?
1) Specimen is placed on metal disc
2) Frozen rapidly to -30 degrees celsius
3) Cryostat (microtome in freezer) cuts specimen
4) Staining
Advantage and disadvantage of using the frozen section technique as opposed to traditional embedding?
Time - frozen section takes 10 minutes, traditional method takes 16 hours
Technical quality of section a lot lower
How does Polarised Light Microscopy work? (3 steps)
1) Light passes through filters then sample - crystalline structure
2) Light emerges from the other side vibrating in many planes and with many wavelengths
3) This forms an image
Clinical relevance of Polarised Light Microscopy?
Detect Gout and Pseudogout as crystals will have precipitated in joints
Difference between Gout and Pseudogout in Polarised Light Microscopy?
Gout
-Yellow needle shaped Monosodium Urate (MSU) crystals
-Most common in toe
Pseudogout
- Blue rhombus shaped Calcium Pyrophosphate Dihydrate (CPPD) crystals
-Most common in knee
How does Fluorescent Microscopy work? (3 steps)
1) Fluorophores attached to sample
2) Irradiate by specific wavelengths causing fluorophores to emit light
3) Different colours appear indicate different cell components
How does Confocal Microscopy work? (2 points)
1) Resembles a Computed Axial Tomography (CAT) scan
2) Computer can look through body in slices without actually slicing it!
How can immunohistochemistry be used as an investigative technique? (3 points)
1) To find out if a specific protein is present in a cell/tissue
2) An antibody against this protein utilised
3) A second antibody added that joins to first and colours when stained
How does Immunofluorescence work?
1) To find out if a tissue/cell has a protein
2) Antibody against this protein added
3) Fluorescent tag added that attaches to first antibody
How does autoradiography work?
1) Radioactive marker injected into live cell
2) Photographic emulsion used to visualise molecules labelled with marker
Clinical relevance of autoradiography?
Iodine 131 can be used to show an enlarged right thyroid lobe as thyroid takes up iodine
If we want to look at cell components using Modern Light Microscopy, what do we have to change about the light used?
Waves with much smaller wavelength must be used
