Microscopy Flashcards
To understand how different types of microscopes function and how to use them
How do you calculate magnification?
magnification= size of image/ size of real object.
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What are the features of an optical microscope?
They use light to form an image
They have a maximum resolution of 0.2 micrometres.
The maximum magnification is about x 1500
What are the features of an electron microscope?
They use electrons to form an image
They have a higher resolution than optical microscopes- so give a more detailed image.
They have a maximum resolution of 0.0002 micrometres. This is about 1000 times higher than optical microscopes.
The maximum resolution is about x 1 500 000
What are the features of a transmission electron microscope (TEM)?
TEMs use electromagnets to focus a beam of electrons, which is transmitted through the specimen.
Denser parts of the organism absorb more electrons, which makes them look darker on an image.
TEMs give higher resolution images, so you see the internal structure of organelles
Can only be used on thin specimens.
What are the features of a scanning electron microscope (SEM)?
SEMs scan a beam of electrons across the specimen. This knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image
The image you end up with show the surface of the specimen and they can be 3D
Can be used on thick specimens
Give lower resolution images than TEMs
How to prepare a slide for microscopic study:
Start by pipetting a small drop of water onto the slide.
Then use tweezers to place a thin section of the specimen on top of the water.
Add a drop of stain. The stain is used to highlight objects in a cell.
Add the cover slip Stand the clip upright on the slide, next to the water droplet. Then carefully lift and lower it so it covers the specimen.
Don’t get air bubbles.
What is cell fractionation and how does it work?
Cell fractionation- separating the organelle you want to analyse from the rest of the cell.
What is homogenisation in cell fractionation?
Homogenisation (breaking up the cells)- Can be done in several different ways, e.g vibrating the cells or by grinding the cells up in a blender.
This breaks up the plasma membrane and releases the organelles into solution. Solution must be kept ice cold, to reduce the activity of enzymes that break down organelles. Solution should also be isotonic, means it should have the same concentration of chemicals as the cells being broken down to prevent damage to the organelles through osmosis. A buffer solution should be added to maintain pH..
What is fractionation in cell fractionation?
Filtration (getting rid of the bits)- homogenised cell solution is filtered through a gauze to separate any large cell debris or tissue debris.
What is ultracentrifugation in cell fractionation?
Ultracentrifugation (separating the organelles)- Cell fragments are poured into a tube. The tube is put into a centrifuge and is spun at a low speed. The heaviest organelles, like nuclei, get flung to the bottom of the tube by the centrifuge. They form a thick sediment at the bottom (the pellet). The rest of the organelles stay suspended in the fluid above the sediment (the supernatant)
The supernatant is drained off, poured into another tube and spun in the centrifuge at a higher speed. Again, the heaviest organelles, this time the mitochondria, form a pellet at the bottom of the tube.
The supernatant containing the rest of the organelles is drained off and spun in the centrifuge at an even higher speed.
This process is repeated at higher and higher speeds until all the organelles are separated/.
How to use a microscope to observe cells?
Clip the slide you’ve prepared onto the slide
Select the lowest-powered objective lens
Use the coarse adjustment knob to bring the stage up to just below the objective lens
Look down the eyepiece. Use the coarse adjustment knob to move the stage downwards away from the objective lens until the image is roughly in focus.
Adjust the focus with the fine adjustment knob until you get a clear image.
Greater magnification= a higher powered objective lens
