3.2.1.3 methods of studying cells Flashcards

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1
Q

describe the method to calculate mean number of organelles using an optical microscope.

A
  1. measure diameter of field of view and calculate area
  2. using stage micrometer and eyepiece graticule
  3. count the number of capillaries in large number of fields of view and calculate mean
  4. select fields of view randomly
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2
Q

What is resolution?

A

+ how much detail the image has; the better the resolution is the microscope is able to distinguish between two points that are close together

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3
Q

What is magnification?

A

+ how much bigger the image is compared to the specimen sample

magnification = image size/actual size

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4
Q

What is an optical microscope?

A

+ uses light to form the image and focuses this light using glass lenses
+ maximum resolution 0.2µm
+ cannot view smaller organisms such as; ribosomes, ER and lysosomes, mitochondria may be visible but not clear
+ maximum magnification is approx x1500

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5
Q

How do you prepare a temporary mount?

A
  1. pipette a drop water onto the slide (so the specimen sticks)
  2. use tweezers to place a thin section of specimen on the damp
  3. add a drop of stain (e.g eosin for cytoplasm or iodine for starch grains)
  4. add a cover slip to protect the specimen (making sure not to get any air bubbles in)
  5. thin (single/few layers of cells) to allow light to pass through the specimen
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6
Q

What are the advantages of optical microscopes?

A

+ can see living specimens
+ cheaper
+ show colour
+ less time consuming to prepare slides

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7
Q

What are the disadvantages of optical microscopes?

A

+ lower magnification and resolution
+ 2D
+ can’t view smaller organelles in detail

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8
Q

What is a TEM?

A

+ transmission electron microscope
+ specimen must be very thin and placed in a vacuum
+ a beam of electron passes through the specimen and is dispersed by the structures there
+ scattered electrons are then captured on a photographic plate
+ electrons pass through denser part of sample less easily so areas that are darker are more denser
+ 2D
+ magnification up to x250 000
+ resolution 2nm

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9
Q

What are the advantages of electron microscopes?

A

+ can have a x200 better resolution that light microscopes - because electrons have a shorter wavelength
+ produced detailed images
+ SEM produces 3D images

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9
Q

What is SEM?

A

+ electrons ‘bounce’ off metal-salt-stained sample
+ uses an electromagnet to focus the beam of electrons
+ 3D image is built up on a screen
+ magnification x100 000
+ resolution between 3 to 20nm

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10
Q

What are the disadvantages of electron microscopes?

A

+ the interior is a vacuum - cannot view live specimens
+ requires very careful staining and the specimen often has to be very thin however image is only black and white
+ can lead to artefacts
+ very expensive

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11
Q

What are some limitations of TEM?

A

+ specimen must be very thin to allow the electrons to penetrate
+ therefore flat 2D image
+ can overcome by taking series of sections

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12
Q

What are some limitations of SEM?

A

+ lower resolution than TEM but still 10 times better than light microscope

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13
Q

What are artefacts?

A

+ things that you can see down the microscope that aren’t part of the specimen you are looking at
+ e.g. dust, air bubbles, fingerprints
+ usually made during the preparation of the specimen

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14
Q

What is ultracentrifugation?

A

+ process where cells are broken up and the different organelles they contain separated out
+ used to help us study cell structure and function
+ two stage process: homogenation and ultracentrifugation

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15
Q

Step one of ultracentrifugation

A

+ tissue to be studied is cut into smaller pieces (minced) and placed in a cold, isotonic and buffered solution

16
Q

Step two of ultracentrifugation

A

+ these are then ground into smaller pieces using a homogensier

17
Q

Step three of ultracentrifugation

A

+ this releases the organelles from within the cell membrane

18
Q

Step four of ultracentrifugation

A

+ the homogenate is filtered to remove any complete cells and large debris

19
Q

Step five of ultracentrifugation

A

+ a suspension of homogenate is then placed in a test tube and then centrifuged
+ the faster the speed at which the tube is spun, the greater the force generated

20
Q

Step six of ultracentrifugation

A

+ at sloer speeds the larger fragments collect at the bottom of the tube and smaller ones remain near the top suspended in a liquid called the SUPERNATENT LIQUID

21
Q

Step seven of ultracentrifugation

A

+ these larger fragments (sediment pellets) are then removed and the supernatent remaining is respun at a faster speed
+ some of the smaller fragments collect at the bottom forming a new pellet

22
Q

What is the order of organelle collection?

A
  1. whole cells, nuclei, cytoskeletons
  2. mitochondria, lysosomes, peroxisomes
  3. microsomes, other small vesicles
  4. ribosomes, viruses, large macromolecules