Lab Quiz 2

Question 1

Microscopy

Answer 1

• Early compound microscope credited to Z. jansen around 1590.
• Later improved by Anton Van Leeuwenhoek in mid 17th century.
• Electron microscopy developed in 1930s

Question 2

Resolution

Answer 2

The ability to discern two distinct points in space.

Question 3

Factor influencing resolution

Answer 3

Particle wavelength (gamma)
Refractive index of the medium (n)
Angular aperture.

Question 4

Characteristics of Light Microscopy

Answer 4

• Can magnify images up to about 1400x
• Uses visible light for illumination
• Resolution down to ~200nm (w/ oil)
• Objects ~500 nm can be studied (bacteria, mitochondria)

Question 5

Advantages of light microscopy

Answer 5

• Inexpensive
• easy preparation
• Can use living cells

Question 6

Disadvantages of light microscopy

Answer 6

• Lower resolution
• difficult to see translucent cells

Question 7

Microscopy methodology (light microscopy)

Answer 7

• Sometimes, tissues are fixed to prevent cell degradation.
• Tissues are embedded to allow slicing
• Sections are cut using a microtone

Question 8

Types of Light Microscopy

Answer 8

• Bright-field microscopy - normal white light is passed through a specimen, light is absorbed (or transmitted) and the image is based on light.
• Phase-contrast microscopy - refracts light differently as it travels at different rates through materials of different composition
• Differential Interference contrast (DIC) - polarized light passes through material differently due to different index of refraction.

Question 9

Fluorescence

Answer 9

fluorochromes are molecules that are excited by light at one wavelength and then emit light at a difference wavelength.

(special dyes attached to abs can identify individual molecules within living cells).

Question 10

Fluorescence microscopy

Answer 10

excites a chemical at one wavelength that fluoresces back at a separate, longer wavelength.

Question 11

Green fluorescent protein (GFP) labeling

Answer 11

• A common fluorochrome
• Gene for fluorescent protein can be inserted into a cell’s genome.

Question 12

Indirect immunocytochemistry

Answer 12

use of secondary abs to generate immunofluorescence.

Question 13

Confocal Microscopy

Answer 13

• Light from a laser is passed through a confocal pinhole onto the specimen
• Fluorescence from the specimen is passed to the detector through a second pinhole.
• The pinhole blocks out the blurred light emitted from areas that are different plane focus.

(Blurring from light out of the plane of focus reduces resolution.
If the light from the layers are not in the plane of focus is removed)

Question 14

Electron Microscopy

Answer 14

• Uses electron beams rather than light beams to inimunate the specimen.
• electron wavelength is very small leading to a higher resolving power (0.1 -2.0 nm)

Question 15

Two types of Electron Microscopy commonly used:

Answer 15

1. Transmission electron microscopy (TEM): resolution 0.1 - 2.0 nm
2. Scanning electron microscopy (SEM): resolution 3 - 10nm.

Question 16

TEM (traditional)

Answer 16

• Sliced specimen is stained with electron-dense material (heavy metals).
• Electrons projected through a vacuum to strike the specimen.
• Electrons striking the stained material are scattered and not collected.
• Electrons that are transmitted through

Question 17

SEM

Answer 17

• Electrons strike the surface and are scattered based on surface angles.
• or absorbed energy causes the release or a secondary electron.
• electrons scatter is measure by detector to create surface image.
• SEM has less resolution than TEM
• TEM has to be sliced very thin
• Negative of electron microscopy