Microscopy and Cell fractionation Flashcards
(29 cards)
What is field of view?
lowest magnification means you can see more cells
What does resolution mean?
ability to distinguish between two different but adjacent objects
What does magnification mean?
how many times an object has been enlarged
What is the formula for total magnification?
Total magnification: eyepiece lens x objective lens
What is the magnification formula?
Real size = image size/magnification
What are electron microscopes?
negatively charged electrons can be focused using electromagnets
how does the electron beam compare to light microscopes?
short wavelength compared to light; so, it has a higher resolving power
What are the two types of electron microscopes?
Scanning and transmission
How to TEM’s work?
use electromagnets to focus a beam of electrons
- Denser parts absorb more electrons (appear darker on the final image)
What are advantages of TEM’s?
- High resolution
- Can see internal structures
What are disadvantages of TEM’s?
- Only thin specimens can be used
- the specimen cannot be alive
- Lengthy preparation
- Artefacts
What are artefacts?
look like real structures but are the result of preparation and staining of a sample
How do SEM’s work?
beam of electrons fired across the specimen (produces a 3D image)
What kind of image are SEM’s?
3D
What are the advantages of SEM’s?
- Can be used for thick or 3D
- Allow external structure to be shown
What are the disadvantages of SEM’s?
- Lower resolution than TEMs
- Don’t provide a colour image
- No live specimens
Why is the light microscope resolution poor?
wavelength of light is longer
What can light microscopes see?
Eukaryotes, mitochondria and chloroplasts
What is max magnification of light?
x1500
What is stage 1 of cell fractionation?
Placing in a cold, isotonic buffer solution
Homoginisation - breaking the cells up
What are the three stages of cell fractionation?
Homogenisation
Filtration
Ultracentrifugation
Why are the cells placed in a cold isotonic buffer solution?
Cold - reduce enzyme activity
isotonic - prevents water from moving in cells
Buffer - maintains the pH
What is stage 2 of fractionation?
filtered through a sieve to remove insoluble material
What is stage 3 of fractionation?
ultracentrifugation - spun first at low speed then again at higher speed
