Microscopy

Question 1

What size does an object have to be in order to be visualized?

Answer 1

1/2 the wavelength of the electromagnetic radiation being shown on it

Question 2

What is resolution?

Answer 2

resolution refers to the ability to distinguish two points from each other. Smaller resolution = better

Question 3

What is the wavelength of a light microscope?

Answer 3

0.2 um

Question 4

What is the wavelength of electron microscopy?

Answer 4

2 nm

Question 5

How can you improve resolution?

Answer 5

1. change the illumination source (electrons)
2. increase the intensity of the illumination
3. replace the medium light travel through (prevent refraction)

Question 6

What oil is used for 100x objective?

Answer 6

immersion oil

Question 7

What is contrast?

Answer 7

how distinct is the specimen from the background

Question 8

How can you improve contrast?

Answer 8

1. Positive stain (specimen)
2. Negative stain (background)

Question 9

What are the different types of light microscopy?

Answer 9

1. Bright field
2. Darkfield
3. phase contrast
4. Interference
5. Fluorescence
6. “Superresolution” fluorescence
7. Confocal scanning laser

Question 10

How does brightfield work?

Answer 10

Uses light, diaphragm, and condenser to condense light through slide.

Question 11

How does darkfield work?

Answer 11

Uses a special type of condenser to bend light away from objective. If light hits specimen, it will be bent up into lens

Question 12

What is phase contrast?

Answer 12

Uses phase-shifting of refracted light (caused by photon drag) to enhance contrast. refractive index differences are amplified

Question 13

What is interference microscope?

Answer 13

uses 2 beams of 90 degree polarized light to detect 3D details in the sample. light is separated by prism and recombine after specimen.

Question 14

What is fluorescence microscope?

Answer 14

Uses excitation wavelength as illumination light, can see emission wavelength of fluorescently-labeled specimen

Question 15

What are fluorophores?

Answer 15

chemicals that emit visible light when they are illuminated by a shorter wavelength of light (usually UV)

Question 16

What is superresolution fluorescence?

Answer 16

uses very brief excitation of low-intensity fluorophore stain, followed by computer averaging, to enhance resolution of light microscope

Question 17

Can you use multiple fluorophores?

Answer 17

Yes, they are linked to different antibodies

Question 18

What is confocal scanning laser microscope?

Answer 18

uses an aperture to shut out light from all but one focal plane of the object. Computer builds up 3-d object plane by plane

Question 19

Types of electron microscopy?

Answer 19

1. TEM
2. SEM
3. electron cyrotomography

Question 20

What is TEM?

Answer 20

transmission electron microscopy; electron beam passes through the specimen, just like a beam of light. uses dye that absorbs electrons

Question 21

What is SEM?

Answer 21

Scanning electron microscopy; detects electrons and x-rays scattered from a surface, dramatic 3D views

Question 22

What is electron cryotomography?

Answer 22

rotate object; capture images at different angles. use software to compile a 3d shape

Question 23

What is atomic force microscopy?

Answer 23

electric field is monitored at the tip of a nanotube probe. Field varies as probe is pushed over a surface. Can see atoms

Question 24

What is a simple stain?

Answer 24

shows outline of bacteria; basic (+) dye attaches to (-) phospholipid head groups?

Question 25

What is differential stain?

Answer 25

more than one stain is used - cell stain differently based on call properties. Includes gram stain and acid-fast stain

Question 26

What is a gram stain?

Answer 26

shows thickness of peptidoglycan; more than one stain is used

Question 27

What are gram stain results?

Answer 27

pink = thin (gram -)
purple = thick (gram +)

Question 28

What is acid-fast stain?

Answer 28

used for bacteria with waxy cell walls; differentiates based on waxy coating impervious to acidified alcohol

Question 29

What are acid-fast stain results?

Answer 29

red = acid-fast (waxy)
blue = not

Question 30

What is negative stain?

Answer 30

stains the background, not the cell