Microscopy Flashcards
Why is cell fractionation needed
- Needed to study the structure and function of various organelles that make up a cell
- we need a large number of isolated organelles which we can gate from cell fractionation
Define cell fractionation
The process in which cells are brocken up and the different organelle they contain are separated out
Describe the process of homogenisation
- tissue place in cold Isotonic, buffered solution
- cells Brocken up by a homogeniser/blender
- breaks up cells in tissue, releases organelles from cells
- homogenate is filled to remove complete cells and large debris
Why is isotonic, buffered solution cold
To reduce enzyme activity’s that could break down organelles
Eg lysozymes
Why is isotonic, buffered solution buffered
- so pH doesn’t fluctuate
- change in pH could alter the structure of organelles affecting the functioning enzymes
Why is isotonic, buffered solution have the same water potential as the tissue
- Prevent organelles from bursting or shrinking as a result of osmotic gain or loss of water
Principle of ultracentrifugation
Heavy organelles will fall to the bottom first in the sediment leaving the clear supernatve which will be removed and re spun for loner and faster
What would u find in sediment 1
Heaviest organelles eg nuclei
What happens to supernatent 1
Taken and re-spun at medium speed
What would u find in sediment 2
Next heaviest organelles eg mitochondria
What happens to supernatent 2
Taken and spun at high speed
What would u find in sediment 3
Contains next heaviest organelles eg lysosomes
How do u get greater resolution
Shorter wavelengths
What does a greater resolution do to the image
Make it the image clearer
Define resolution
Minimum distance apart 2 objects can be in order for them to appear separate
What happens after resolution reaches it limit
Image gets larger and blurred
Define magnification
Ho many times bigger is the image than the actual object
Method of using a light microscope
- add a drop of water to a glass slide
- obtain a thin section of specimen and place on slide
- stain with iodine of potassium iodide
- lower cover slip using a mounted needle
Why when using a light microscope must the specimen be stained
Thin section are transparent staining them means you can see them
Why are specimen thin on a light microscope
So light can pass through the specimen and so you can only focus of one layer of cell
Weaknesses o a light microscope
- Poor resolution
- very little intercellular detail can be seen
Poor resolution
Long wavelength of light
What are the 2 types of electron microscope
- TEM
- SEM
Differences between light and electron microscopes
- uses a beam of electrons rather than light
- electron microscope has higher resolution power whereas light microscope has poor resolution
- electron has shorter wavelength whereas light has long wavelengths
Why a electron beam can be focused more precisely than a light beam
electrons are negatively charged some o can be o used through a electromagnets whereas light has no charge
What TEM microscope specimen stained with
Stains containing heavy metals
2 advantages of using a transmission electron microscope
- high resolution images are produced
- small objects can be displayed
2 disadvantage of using a transmission electron microscope
- can only be used of thin specimens
- microscope only works on non living specimens
Give 2 disadvantage of a scanning electron microscope
- lower resolution images given
- only can be used on living specimens
Why is an image consisting of bright and dark areas produced from a TEM
The specimen is very thin and will allow some electrons to penetrate but not others this results in the detection of light and dark areas
Why can’t we use electron microscope to view live specimens
Because the entire process takes place in a vacuum. This is because electrons are absorbed or deflected by molecules of air, and this would prevent them from reaching the specimen
How does a SEM work
Scanning electron microscopes fire electrons across the surface of the specimen to be reflected back and forth
Wha type of image does a SEM produce
- 3D image
- image primary black and white but colour can be added by a computer
Similarities between SEM and TEM
- Better than light microscopes
- both may contain artefacts
- beam of electrons used
- vacuum
- complex staining process
Differences between SEM and TEM
- specimens don’t need to be as thin in SEM
- SEM produces 3D images whereas TE produces 2D images
- SEM = X 100 000 TEM = X 500 000
- SEM = 20nm TEM = 0.1nm
How do u calibrate a graticule
- Use a stage micrometer
- line up scales on graticule and micrometer
- then calculate length of divisions on eyepiece graticule
Why are graticule needed
- needed to measure size of objects under objective lens
- you need to calibrate the graticule as each objective will magnify to a different degree
