Microscopy

Question 1

What are the types of light microscopy?

Answer 1

There’s 5
1. bright-field
2. phase contrast
3. DIC (differential interference contrast)
4. Dark field (scattered light)
5. Fluorescence

Question 1

What type of electron microscopy are there?

Answer 1

There’s 2:
1. Transmission electron microscopy
2. Scanning electron microscopy

Question 2

What is brightfield?

Answer 2

it is light microscopy that takes advantage of staining

Question 3

What are the types of staining for brightfield?

Answer 3

• Crystal violet
• Carbol fuchsin
• Methylene blue
• Negative staining
• Flagellar stain
• Endospore stain
• Capsule stain
• Gram staining

Question 4

What is gram staining and what are the steps to it?

Answer 4

Gram staining works due to the different cell envelopes that the cells have
- there’s gram positive and gram negative cells and gram positive cells are able to retain the stains
the staining is done in 5 steps
1. Fixation
- Helps stick the cell to the glass
2. Crystal violet
- Colors the cell blue
3. Iodine (mordant)
4. Decolorization (ethanol)
- Gram negative cells would be clear
- Gram positive cells would be purple
5. Counterstain with safranin
- Gram negative cell would be pink
- Gram positive cells would be blue-purple

Question 5

What is an example of a gram positive and gram negative cell

Answer 5

gram positive: Staphylococcus
gram negative: E.coli

Question 6

Phase contrast

Answer 6

Allows one to see transparent cell (so no staining required)
when light passes through the specimen, the speed is slowed and the phase is delayed and the light ray bends (refraction)
so this causes the light to be out of phase with the other light waves
the lights are half a wavelength out of sync from each other and this is destructive interference so cells appear dark

Question 7

what is DIC

Answer 7

differential interference contrast
it uses polarized light and allows us to view things while they are still alive

uses gradients in the optical path length and phase shifts of light to make phase objects visible when viewed with a microscope

Question 8

What is dark field?

Answer 8

allows you to see things that you might not without it
it enhances shapes
it has better light resolution than light microscopes

Question 9

what is fluorescence

Answer 9

the cells are labeled with fluorophore
Illuminated with light of a shorter wavelength
The fluorophore emits light of a longer wavelength
- And since you cant lose energy it will explode and emit as longer wavelength
So the fluorophore binds to the cell directly and antibody conjugated to the fluoropore so genetically engineered cells to express a fluorescent protein fusion

Question 10

transmission electron microscopy

Answer 10

Electrons are detected and they are focused by magnets
They spray the cells with heavy metal ions that allow the electron to bounce off
It reveals differences in density
They look 2-dimensional or flat

Question 11

Scanning electron microscopy

Answer 11

Scattered electrons are detected
Specimen look 3-dimensional

Question 12

True or false? Scanning electron microscopy produces images that are flatter (more 2 dimensional) than transmission electron microscopy?

Answer 12

False

Question 13

True or false? electron microscopes are more powerful than light microscopes because they have greater limit of resolution

Answer 13

false

Question 14

True or false? With a light microscope, you can see the flagella of living bacteria

Answer 14

false, you can only see non-living bacteria

Question 15

True or false? Darkfield microscopy relies upon a negative stain

Answer 15

false

Question 16

True or false? fluorescence microscopy detects light that has travelled through the specimen

Answer 16

false

Question 17

True or false? electrons have a longer wavelength than rays of visible light

Answer 17

false, because electrons have a shorter wavelength than light

Question 18

True or false? DIC microscopy relies upon polarized light

Answer 18

true

Question 19

True or false? phase contrast microscopy causes cells to look like bright objects against a grey background

Answer 19

false, it looks darker not brighter

Question 20

Fill in the blank:
On a compound microscope, the light travels from the bulb, up through two ___ to the specimen on the stage. From their it proceeds to the ____ lens, and then to the ____ lens.

Answer 20

On a compound microscope, the light travels from the bulb, up through two condenser to the specimen on the stage. From their it proceeds to the objective lens, and then to the optic lens.

Question 21

The term describing light bending as it passes through different material is

Answer 21

refraction

Question 22

Fill in the blank:
During the gram stain procedure, just before adding the counterstain, the gram positive cells are ___ and the gram negative cells are ____

Answer 22

During the gram stain procedure, just before adding the counterstain, the gram positive cells are purple and the gram negative cells are clear/white

Question 23

Darkfield microscopy is different from brightfield, because of what two features of the light rays?

Answer 23

1. lights come from the side and angle, shines obliquely
2. light scattering