Methods of Studying Cells Flashcards
what is an object?
material put under a microscope
what is an image?
the appearance of the object when viewed under a microscope
magnification =
size of image divided by size of object
resolution definition
the minimum distance apart that two objects can be for them to appear as separate items under a microscope
what does the resolving power of a microscope depend on?
the wavelength or form of radiation used
what does a greater resolution produce?
a clearer and more precise image
does increasing magnification always increase the resolution?
no - there is a limit
what happens if you increase the magnification past the limit of the resolution?
the image will get larger but more blurry
what is homogenisation?
breaking up cells in a blender (homogeniser) to release the organelles
what is another name for the resultant fluid of homogenisation?
homogenate
what happens to the homogenate?
it is filtered to remove complete cells and unwanted debris
ultracentrifugation definition
the process by which fragments in a filtered homogenate are separated in a centrifuge
what is the importance of cell fractionation?
obtaining large numbers of isolated organelles allows scientists to study their structure and function
cell fractionation step 1
place tissue in a cold, buffered, isotonic solution
why is the solution cold?
to reduce enzyme activity that might break down the organelles
what is isotonic?
same water potential
why is the solution isotonic?
to prevent organelles bursting/shrinking due to osmotic gain/loss of water
why is the solution buffered?
to maintain pH to retain the integrity of the organelles
cell fractionation step 2
homogenise the solution
cell fractionation step 3 (and the first step of ultracentrifugation)
spin the homogenised tissue in a centrifuge at a low speed (1000 X gravity) for 10 minutes. Filter the homogenate to remove unwanted debris.
what does a centrifuge create?
a centrifugal force
cell fractionation step 4 (and the second step of ultracentrifugation)
spin the supernatant (left over) at a medium speed (3500 X gravity). Filter the homogenate to remove unwanted debris.
cell fractionation step 5 (and the third step of ultracentrifugation)
spin the new supernatant at a high speed (16500 X gravity)
what is removed at each increase in speed during ultracentrifugation?
the next heaviest organelles
what is the heaviest organelle?
nucleus
what is the lightest organelle?
ribosomes
what is the second heaviest organelle?
chloroplasts
what is the second lightest organelle?
ER
what is the third heaviest organelle?
mitochondria
what is the third lightest organelle?
lysosomes
how does an optical microscope work?
light is sent from a light source through a specimen, the image of which is magnified by glass lenses
what was the first type of microscope invented?
the light microscope
what is the most common type of microscope?
the light microscope
why are light microscopes the most common?
cheap, easy to use, can study living cells
what type of image does a light microscope produce?
2D
what is the resolution of a light microscope limited to?
200nm or 0.2 micrometers
why is the resolution of a light microscope limited?
light has a relatively long wavelength
what is the magnification of a light microscope limited to?
X 2000
why are stains needed for objects under light microscopes?
individual cells are generally transparent and their components are not distinguishable unless they are coloured with special stains
what is a problem with using stains?
it usually kills the cells
what organelles can you see with a light microscope?
nucleus and mitochondria
what do electron microscopes use instead of light?
a beam of electrons
why do electron microscopes have a higher resolving power?
the electron beam has a shorter wavelength than light
how is the image of an electron microscope produced?
specimens are not directly observed. A computer forms an image based on how many electrons are absorbed by different regions of the specimen
what is the resolution of some of the best electron microscopes?
0.1 nm (2000X more than light)
what are scanning electron microscope images created from?
the electrons that are reflected off the specimen
how do both SEM and TEM work?
directs a beam of electrons at a specimen
how is the beam of electrons focused?
electromagnets
what are the images produced by SEM like?
3D and detailed
do specimens have to be thin when using a SEM?
no
Which has a slightly lower resolution: SEM or TEM?
SEM (1nm)
why are SEM and TEM images black and white?
electrons don’t correspond to light and colour.
Colour can be added artificially to these images
what are some cons to using a SEM or TEM?
expensive
need training to operate it
why must samples be dead for a SEM or TEM?
electrons can be deflected by molecules in the air so a vacuum is needed
how are images from TEM created?
from the electrons that are absorbed by the specimen
how is the magnification of TEM compared to SEM?
2X magnification of SEM
what is the main con of a TEM?
sample must be thin
