Methods of studying cells Flashcards
What is differential centrifugation?
- centrifuging at different speeds (forces)
- based on mass/density of organelles
- used to separate/isolate organelles
What is homogenisation?
Cells are broken open in an ice cold, isotonic, buffer solution using blender/homogeniser
What are the conditions for homogenisation?
- ice cold: reduce enzyme activity
- isotonic: prevents osmotic damage
- pH buffer: prevents denaturing of enzymes/proteins
What is ultracentrifugation?
- (once homogenised) sample filtered and spun at relatively slow speed - most dense organelles form pellet at bottom
- remove supernatent (solution) + spin faster/for longer
- repeat until desired organelle is isolated
What is the general order of density for organelles?
- nuclei
- chloroplasts
- mitochondria
- E.R, Golgi (tend to fragment)
- ribosomes
How to calculate total magnification?
Eyepiece (10) x objective (40 e.g.)
How to calculate magnification
Image size / actual size
Unit conversions
cm (x10) mm (x1000) ųm (x1000) nm
How do light microscopes work?
- Passes light through specimen
- Focused by glass
What are the benefits of light microscopes?
- specimens can be dead or living
- image in colour
- prep is quick + easy
What are the limitations of light microscopes?
Relatively poor resolution (small structures not visible)
Why do light microscopes have a poor resolution?
Light has a longer wavelength
What is magnification?
How many times larger the image is compared to the object
What is resolution?
Minimum distance between 2 objects in which they can still be viewed as separate
Preparation for light microscopy
- thin so light can pass through (single layer is visible)
- stained to make structures visible
How do electron microscopes work?
Beam of electrons focused by electromagnets
What are the benefits of electron microscopes?
greater resolution
Why do electron microscopes have a greater resolution?
Shorter wavelength
What are the limitations of electron microscopes?
- specimen has to be dead + dehydrated
- image not in colour
- prep complex + time-consuming
How does a transmission electron microscope work?
- beam of electrons transmitted through specimen
- specimen must be thin, stained using electron dense substances (e.g. heavy metal salts)
- substances deflect electrons in the beam + pattern remaining electrons produce as they pass through is converted to an image
How does a scanning electron microscope work?
- specimen coated with thin film of heavy metal (e.g. gold)
- electron beam scanned to and fro across specimen
- electrons reflected from surface are collected + produce image on a viewing screen
Characteristics of TEM
- higher resolution than SEM
- produces image of internal structures
- gives 2D image
- sections must be very thin
Characteristics of SEM
- lower resolution than TEM
- produces image of external/surface structures
- gives 3D image
- sections can be thicker than with TEM
