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biol 2321 - cell bio lab > Microscopy & Electron Micrographs > Flashcards

Microscopy & Electron Micrographs Flashcards

1
Q

What illumination source does a bright field microscope have?

A

light

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2
Q

What is the maximum magnification of a BFM?

A

1000x - 2000x total

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3
Q

When are BFMs best used?

A

looking at LIVE specimens

getting an overview of a sample - ex. cell and tissue organization

most economical option

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4
Q

What is a major disadvantage of BFMs?

A

low resolving powers

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5
Q

Define resolution?

A

the smallest distance between two objects at which the observer can distinguish there are two objects

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6
Q

What is the formula for resolution?

A

D = 0.61 lambda/ n sin alpha

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7
Q

Why does resolution increase drastically with the electron microscope?

A

because the illumination source is not light, it is electrons and electrons have a shorter wavelength than light

shorter wavelength = better resolution

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8
Q

When are TEMs best used?

A

for looking at internal structure of cells

for viewing at very high resolution

for looking at relationships between structures

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9
Q

What are the disadvantages of TEMs?

A

samples need extensive preparation before visualizing and specimen must be dead

expensive to run

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10
Q

What is the max magnification of TEMs?

A

5,000,000 x

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11
Q

Describe the structure of a TEM

A

an electron gun (tungsten filament and anode) at the top of the column

electromagnetic condenser lenses throughout column

fluorescent screen and camera at base

specimen holder at base

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12
Q

Describe how a TEM works

A

an electron gun (tungsten filament and anode) directs electrons through electromagnetic rings which focus the electron beam through the specimen lighting up the fluorescent screen at the base

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13
Q

What are the two outcomes of the electron beam moving towards the specimen?

A

either the electrons will hit the stained specimen and reflect off, creating shadows

or the electrons will pass through the specimen and make light dots on the screen

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14
Q

What is the result of a TEM (what the observer sees)?

A

a black and white 2D image with high resolution

a slice of a specimen

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15
Q

In what 3 ways is a TEM similar to a light microscope?

A
  1. its condenser lenses focus beam on specimen
  2. specimen used is SO small (1/4 size of pinky finger nail mounted on grid)
  3. objective, intermediate projection lenses all magnify
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16
Q

How is resolution adjusted on a TEM?

A

by altering the voltage

higher voltage = greater resolution

17
Q

What does increasing the voltage on a TEM do?

A

increase resolution

18
Q

How is magnification adjusted on a TEM?

A

by adjusting the electrical current using the electromagnetic lenses

19
Q

Describe the steps of preparing tissue for TEM?

A
  1. preserve fresh tissue
  2. dehydrate tissue using alcohol series
  3. coat tissue in plastic resin and let harden
  4. Using a microtome with a diamond knife, perform ultrathin sectioning to cut the tissue to 100nm thickness
  5. mount sectioned tissue onto an EM grid for staining
  6. stain tissue with heavy metals that coat the specimen to increase scatter of electrons = more contrast
20
Q

How is an image formed from TEM?

A

some parts of a stained specimen may be denser than others and those dense areas will deflect electrons and create dark spots on the fluorescent screen

Parts that are not as dense will allow electrons to pass through the specimen and create light spots

The result is a 2D slice of the specimen with a black and white contrast image of the specimen

21
Q

Describe how to prepare the specimen for TEM negative staining

A

for outlines of small particulates (ribosomes, isolated Golgi, viruses, multi-subunit enzymes, etc.)

  1. the grid where the specimen will be held is coated with a transparent substance to fill any holes
  2. drops of heavy metal stain are added to the grid and allowed to dry
  3. heavy metal deposits everywhere EXCEPT where the specimen is

result = more contrast between the specimen and the grid

22
Q

Describe how to prepare the specimen for TEM freeze fracture

A
  1. specimen is frozen in liquid nitrogen and cracked with a cold knife to separate parts of the tissue (usually the 2 monolayers of a membrane)
  2. heavy metal is deposited to the frozen membrane pieces
  3. carbon layer deposited directly on top of the metal and acts as a cement/casting
  4. tissue digests/is discarded and the carbon layer can be viewed under TEM

Shows a 3D texture and improves resolution

23
Q

What is the max magnification of a SEM?

A

~100,000x

24
Q

What are SEMs best used for?

A

looking at the surfaces of OBJECTS (not organelles)

larger specimens

looking at objects in 3D

25
Q

What are a few disadvantages of SEMs?

A

resolution is not as high as TEM

specimen must be dead and coated in metal before visualizing

expensive to run

26
Q

How do tissues need to be prepared for viewing in an SEM?

A

tissue must be dehydrated (by sublimation under pressure) but its shape and structures must be maintained

dried specimen must be coated in gold to make it suitable for electron beam

27
Q

Describe how an SEM works

A

an electron beam SCANS the surface of the gold-coated specimen

as the metal evaporates, the atoms condense on the surface of the specimen

electrons are reflected off surface and detected to create a 3D image

28
Q

Which of the 3 microscopes can visualize live specimen?

A

light microscope

29
Q

What kind of lenses do the 3 microscopes use?

A

light: glass

TEM & SEM: magnets

30
Q

What kind of images do each of the 3 microscopes produce?

A

light: coloured images

TEM: 2D black and white slices

SEM: 3D black and white images

31
Q

What kind of stains can be used in each of the microscopes?

A

light: optional coloured stains

TEM: required heavy metal stains to stick to cellular structures

SEM: required gold coating over surface of specimen

32
Q

What is a major advantage of light microscopy?

A

live specimens can be visualized and colour and movement can be observed

33
Q

What is a major advantage of TEM?

A

the best resolving power

34
Q

What is the major advantage of SEM?

A

it produces a 3D image of a specimen

35
Q

What is the benefit of using all 3 microscopes?

A

Looking at the 3 different micrographs will each give us different information about a specimen and combining all 3 will give us a better understanding of the specimen

biol 2321 - cell bio lab (10 decks)

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