Microscopy & Electron Micrographs

Question 1

What illumination source does a bright field microscope have?

Answer 1

light

Question 2

What is the maximum magnification of a BFM?

Answer 2

1000x - 2000x total

Question 3

When are BFMs best used?

Answer 3

looking at LIVE specimens

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

most economical option

Question 4

What is a major disadvantage of BFMs?

Answer 4

low resolving powers

Question 5

Define resolution?

Answer 5

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

Question 6

What is the formula for resolution?

Answer 6

D = 0.61 lambda/ n sin alpha

Question 7

Why does resolution increase drastically with the electron microscope?

Answer 7

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

shorter wavelength = better resolution

Question 8

When are TEMs best used?

Answer 8

for looking at internal structure of cells

for viewing at very high resolution

for looking at relationships between structures

Question 9

What are the disadvantages of TEMs?

Answer 9

samples need extensive preparation before visualizing and specimen must be dead

expensive to run

Question 10

What is the max magnification of TEMs?

Answer 10

5,000,000 x

Question 11

Describe the structure of a TEM

Answer 11

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

Question 12

Describe how a TEM works

Answer 12

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

Question 13

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

Answer 13

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

Question 14

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

Answer 14

a black and white 2D image with high resolution

a slice of a specimen

Question 15

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

Answer 15

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

Question 16

How is resolution adjusted on a TEM?

Answer 16

by altering the voltage

higher voltage = greater resolution

Question 17

What does increasing the voltage on a TEM do?

Answer 17

increase resolution

Question 18

How is magnification adjusted on a TEM?

Answer 18

by adjusting the electrical current using the electromagnetic lenses

Question 19

Describe the steps of preparing tissue for TEM?

Answer 19

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

Question 20

How is an image formed from TEM?

Answer 20

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

Question 21

Describe how to prepare the specimen for TEM negative staining

Answer 21

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

Question 22

Describe how to prepare the specimen for TEM freeze fracture

Answer 22

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

Question 23

What is the max magnification of a SEM?

Answer 23

~100,000x

Question 24

What are SEMs best used for?

Answer 24

looking at the surfaces of OBJECTS (not organelles)

larger specimens

looking at objects in 3D

Question 25

What are a few disadvantages of SEMs?

Answer 25

resolution is not as high as TEM

specimen must be dead and coated in metal before visualizing

expensive to run

Question 26

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

Answer 26

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

Question 27

Describe how an SEM works

Answer 27

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

Question 28

Which of the 3 microscopes can visualize live specimen?

Answer 28

light microscope

Question 29

What kind of lenses do the 3 microscopes use?

Answer 29

light: glass

TEM & SEM: magnets

Question 30

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

Answer 30

light: coloured images

TEM: 2D black and white slices

SEM: 3D black and white images

Question 31

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

Answer 31

light: optional coloured stains

TEM: required heavy metal stains to stick to cellular structures

SEM: required gold coating over surface of specimen

Question 32

What is a major advantage of light microscopy?

Answer 32

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

Question 33

What is a major advantage of TEM?

Answer 33

the best resolving power

Question 34

What is the major advantage of SEM?

Answer 34

it produces a 3D image of a specimen

Question 35

What is the benefit of using all 3 microscopes?

Answer 35

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