Methods of Studying Flashcards
What is resolution?
The minimum distant between two objects where they still can be see as two separate points
What is magnification?
How many times larger the imagine of a specimen observed is compared to the actual size of the specimen
What is the resolution of a light microscope?
200nm
What is the magnification of a light microscope?
x1000
What is an advantage of a light microscope?
it can be used on living cells which means that we can use it to explore processes like cell division
What are the disadvantages of a light microscope?
- a stain is needed which can kill cells
- it has a poor resolution
Why is the resolution of the light microscope poor?
Due to the nature of visible light:
it’s wavelength is too long
Visible light has a wavelength between 400 and 700nm which restricts it’s resolution to 200nm
What is the resolution of an electron microscope?
0.1nm
What is the magnification of an electron microscope?
x1,000,000
What are the two types of electron microscopes?
- Transmission (TEM)
- Scanning (SEM)
How do light microscopes work?
The lens system bends and focuses a beam of visible light that passes through the specimen to produce a magnified image
How do electron microscopes work?
- an electron gun produces a beam of electrons which pass through the microscope
- electromagnetic lenses are used to focus the beam
- the specimen is placed in the path of the beam
- an image appears on a florescent screen
- denser parts of the specimen absorb more electrons which makes them appear darker
What are electromagnetic lenses?
electromagnets located in electron microscopes that are used to focus the beam of electrons
What are the advantage of an electron microscope?
- has a high resolution
- has a high magnification
What are the disadvantage of an electron microscope?
- it can produce artefacts
- the interior is a vacuum so it can’t view living things
- prepping specimen is time consuming as it needs to be stained in a specific way and it needs be extremely thin
What is an artefact?
A false image created through stains or the conditions in the microscope
Why is the inside of an electron microscope a vacuum?
It prevents the beam of electrons from colliding with air particles which could effect the image displayed
What are the key features of a TEM?
- electron beams pass through the specimen
- it produces 2D images
- only works if the specimen is thinly sliced
- it has a very high resolution
What are the key features of a SEM?
- electrons are shattered from the surface of the specimen and detected
- it produces 3D images
- the specimen must be coated with a metal (like gold) which can lead to artifacts
- has a lower resolution than TEM
What are the advantages of using a TEM microscope?
- it has a high resolutions
- you can see small internal organelles
Why are not all organelles included in images?
- they are not stained
- in another part of the cell that the scientist isn’t examining
How do you prepare a slide to see specific molecules?
- Add drop of water to the glass slide
- Then obtain a thin sample of the specimen (1 cell thick)
- Gently place it on the slide and smooth it out to remove any air bubbles
- Add a drop of stain depending on the molecule (starch would need iodine)
- Finally lower the cover slip on with a mounted needle
Why might dark lines appear when staining a cell membrane?
The stain binds to:
* the glycerol molecules
* the phosphate heads of the phospholipids in/outside the cell membrane’s bilayer
What is Cell Centrifugation?
The process in which different parts and organelles of a cell a separated so that they can be studied in detail
What is the homogenate?
A resultant fluid made up of blended tissue
How is centrifugation carried out?
- the tissue is blended and filtered
- then it is places in a centrifuge
- it is then spun at a low speed which pulls the heavier organelles into the pellet (bottom) and the rest into the supernatant
- The supernatant is then removed and placed in a new centrifuge to be spun again at a higher speed
- This is then repeating, increasing the speed each time
Why is the tissue blended?
to open up the cells
Why is the tissue filtered
to remove any unbroken cell/tissue debris
What organelle is in the pellet when the speed is the lowest (800g or spun for 10 minutes)?
Nuclei
What organelle is in the pellet when the speed is 20,000g (or spun for 15 minutes)?
Mitochondrion (and Chloroplast)
What organelle is in the pellet when the speed is 100,000g (or spun for 1 hour)
plasma and membranes (microsomes)
What organelle is in the pellet when the speed is 150,000g (or spun for 3 hours)
Ribosomes
What are the key feature of the solution the homogenate is added to must be?
- contain a buffer solution
- the buffer solution must have the same water potential as the cells (isotonic)
- cold
What is the purpose of a buffer solution in the homogenate?
It maintains the solution’s pH. This important as if the pH changes then the enzymes in the cells could denature
Why should the buffer solution have the same water potential as the cells?
So water doesn’t entre the cells through osmosis and burst/destroy the cells
Why must the homogenate solution be cold?
It prevents any destructive enzymes damaging the organelles by making them inactive
why do specimen on a light microscope have to be thin?
to allow more light can pass through and so one layer of cells can be viewed
